Droplet Actuator Devices and Methods Employing Magnetic Beads

ABSTRACT

A method of providing a droplet in contact with a magnetically responsive bead and having a reduced quantity of a substance. The method generally includes the steps of (a) providing a droplet actuator ( 200 ) comprising: (i) a substrate ( 210 ) comprising electrodes ( 214 ) arranged for conducting droplet operations on a surface; (ii) a starting droplet ( 218/222 ) comprising: (1) one or more magnetically responsive beads ( 220 ); (2) a starting quantity of the substance; and (3) a starting volume; (b) magnetically immobilizing the one or more magnetically responsive beads at a location which is at a distance from a target droplet splitting zone, ( 224 ); (c) conducting one or more droplet operations comprising droplet dividing operations selected to divide the combined droplet to yield a set of droplets comprising: (i) a droplet ( 218 ) comprising substantially all of the one or more magnetically responsive beads and having a decreased quantity of the substance relative to the starting concentration; and (ii) a droplet ( 222 ) substantially lacking the magnetically responsive beads.

1 RELATED APPLICATIONS

This application claims priority to and incorporates by reference related provisional U.S. Patent Application No. 60/900,653, filed on Feb. 9, 2007, entitled “Immobilization of magnetically-responsive beads during droplet operations”; 60/980,772, filed on Oct. 17, 2007, entitled “Immobilization of magnetically-responsive beads in droplets”; 60/969,736, filed on Sep. 4, 2007, entitled “Droplet actuator assay improvements”; and 60/980,762, filed on Oct. 17, 2007, entitled “Droplet actuator assay improvements.”

2 GRANT INFORMATION

This invention was made with government support under CA114993-01A2 and DK066956-02 awarded by the National Institutes of Health of the United States. The United States Government has certain rights in the invention.

3 FIELD OF THE INVENTION

The invention relates generally to the field of droplet actuators, and in particular, to droplet actuators configured for conducting droplet based protocols requiring droplet operations to be conducted using droplets comprising beads, especially magnetically responsive beads. The invention also relates to methods of making and using such droplet actuators.

4 BACKGROUND OF THE INVENTION

Droplet actuators are used to conduct a wide variety of droplet operations. A droplet actuator typically includes a substrate associated with electrodes for conducting droplet operations on a droplet operations surface thereof and may also include a second substrate arranged in a generally parallel fashion in relation to the droplet operations surface to form a gap in which droplet operations are effected. The gap is typically filled with a filler fluid that is immiscible with the fluid that is to be subjected to droplet operations on the droplet actuator.

In some applications of a droplet actuator there is a need for using “beads” for various assays. For protocols that make use of beads, the beads are typically used to bind to one or more target substances in a mixture of substances. The target substances may, for example, be analytes or contaminants. There is a need for an efficient approach to bead washing on a droplet actuator in order to reduce the amount of one or more substances in a bead-containing droplet that may be in contact with or exposed to the surface of the bead or beads.

5 BRIEF DESCRIPTION OF THE INVENTION

One aspect of the invention provides efficient immobilization of magnetically responsive beads for droplet operations that make use of magnetically responsive beads in droplet-based applications. Examples include assays that require execution of bead washing protocols, such as pyrosequencing and immunoassay applications. In one example, the invention provides techniques employing magnetic forces for immobilizing magnetically responsive beads during droplet splitting operations. The techniques of the invention are particularly useful in protocols for washing magnetically responsive beads within a droplet actuator. Among other advantages, the techniques of the invention avoid undue clumping or aggregation of the magnetically responsive beads. During droplet splitting operations, the techniques can usefully immobilize substantially all of the magnetically responsive beads within a droplet. Techniques of the invention may ensure immobilization and retention of substantially all magnetically responsive beads during a droplet washing operation. Upon completion of a washing process, the techniques of the invention ensure resuspension of substantially all of the magnetically responsive beads within the liquid and with substantially no clumping or aggregation thereof.

Another aspect of the invention provides improved droplet actuators and related methods for effecting improved droplet-based assay operations. Embodiments of the invention provide mechanisms for reducing the crossover of magnetic fields within a droplet actuator. Other embodiments of the invention provide mechanisms for reducing the carryover of beads and other substances within a droplet actuator. Yet other embodiments of the invention provide mechanisms for improving droplet detection operations within a droplet actuator.

6 DEFINITIONS

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting a change in the electrical state of the one or more electrodes which results in a droplet operation.

“Bead,” with respect to beads on a droplet actuator, means any bead or particle that is capable of interacting with a droplet on or in proximity with a droplet actuator. Beads may be any of a wide variety of shapes, such as spherical, generally spherical, egg shaped, disc shaped, cubical and other three dimensional shapes. The bead may, for example, be capable of being transported in a droplet on a droplet actuator or otherwise configured with respect to a droplet actuator in a manner which permits a droplet on the droplet actuator to be brought into contact with the bead, on the droplet actuator and/or off the droplet actuator. Beads may be manufactured using a wide variety of materials, including for example, resins, and polymers. The beads may be any suitable size, including for example, microbeads, microparticles, nanobeads and nanoparticles. In some cases, beads are magnetically responsive; in other cases beads are not significantly magnetically responsive. For magnetically responsive beads, the magnetically responsive material may constitute substantially all of a bead or one component only of a bead. The remainder of the bead may include, among other things, polymeric material, coatings, and moieties which permit attachment of an assay reagent. Examples of suitable magnetically responsive beads are described in U.S. Patent Publication No. 2005-0260686, entitled, “Multiplex flow assays preferably with magnetic particles as solid phase,” published on Nov. 24, 2005, the entire disclosure of which is incorporated herein by reference for its teaching concerning magnetically responsive materials and beads. The beads may include one or more populations of biological cells adhered thereto. In some cases, the biological cells are a substantially pure population. In other cases, the biological cells include different cell populations, e.g., cell populations which interact with one another.

“Droplet” means a volume of liquid on a droplet actuator that is at least partially bounded by filler fluid. For example, a droplet may be completely surrounded by filler fluid or may be bounded by filler fluid and one or more surfaces of the droplet actuator. Droplets may take a wide variety of shapes; nonlimiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more surfaces of a droplet actuator.

“Droplet operation” means any manipulation of a droplet on a droplet actuator. A droplet operation may, for example, include: loading a droplet into the droplet actuator; dispensing one or more droplets from a source droplet; splitting, separating or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing. The terms “merge,” “merging,” “combine,” “combining” and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, “merging droplet A with droplet B,” can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms “splitting,” “separating” and “dividing” are not intended to imply any particular outcome with respect to size of the resulting droplets (i.e., the size of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term “mixing” refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of “loading” droplet operations include microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading.

“Immobilize” with respect to magnetically responsive beads, means that the beads are substantially restrained in position in a droplet or in filler fluid on a droplet actuator. For example, in one embodiment, immobilized beads are sufficiently restrained in position to permit execution of a splitting operation on a droplet, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads.

“Magnetically responsive” means responsive to a magnetic field. “Magnetically responsive beads” include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe₃O₄, BaFe₁₂O₁₉, CoO, NiO, Mn₂O₃, Cr₂O₃, and CoMnP.

“Washing” with respect to washing a magnetically responsive bead means reducing the amount and/or concentration of one or more substances in contact with the magnetically responsive bead or exposed to the magnetically responsive bead from a droplet in contact with the magnetically responsive bead. The reduction in the amount and/or concentration of the substance may be partial, substantially complete, or even complete. The substance may be any of a wide variety of substances; examples include target substances for further analysis, and unwanted substances, such as components of a sample, contaminants, and/or excess reagent. In some embodiments, a washing operation begins with a starting droplet in contact with a magnetically responsive bead, where the droplet includes an initial amount and initial concentration of a substance. The washing operation may proceed using a variety of droplet operations. The washing operation may yield a droplet including the magnetically responsive bead, where the droplet has a total amount and/or concentration of the substance which is less than the initial amount and/or concentration of the substance. Other embodiments are described elsewhere herein, and still others will be immediately apparent in view of the present disclosure.

The terms “top” and “bottom” are used throughout the description with reference to the top and bottom substrates of the droplet actuator for convenience only, since the droplet actuator is functional regardless of its position in space.

When a given component, such as a layer, region or substrate, is referred to herein as being disposed or formed “on” another component, that given component can be directly on the other component or, alternatively, intervening components (for example, one or more coatings, layers, interlayers, electrodes or contacts) can also be present. It will be further understood that the terms “disposed on” and “formed on” are used interchangeably to describe how a given component is positioned or situated in relation to another component. Hence, the terms “disposed on” and “formed on” are not intended to introduce any limitations relating to particular methods of material transport, deposition, or fabrication.

When a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being “on”, “at”, or “over” an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a droplet actuator, it should be understood that the droplet is arranged on the droplet actuator in a manner which facilitates using the droplet actuator to conduct one or more droplet operations on the droplet, the droplet is arranged on the droplet actuator in a manner which facilitates sensing of a property of or a signal from the droplet, and/or the droplet has been subjected to a droplet operation on the droplet actuator.

7 BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate first and second top views, respectively, of a portion of a droplet actuator in use during a first and second phase, respectively, of a droplet splitting operation;

FIGS. 2A and 2B illustrate a side and top views of a section of a droplet actuator that includes a magnet that is placed at a position which is away from the splitting zone;

FIG. 3 illustrates a side view of a section of a droplet actuator that includes two magnets that are arranged above and below droplet;

FIGS. 4A and 4B illustrate side views of a section of a droplet actuator that includes four magnets that are arranged at positions surrounding the droplet;

FIGS. 5A and 5B illustrate a first and second top view, respectively, of a section of a droplet actuator during a first and second phase, respectively, of a droplet splitting operation;

FIG. 6 illustrates a side view of a section of a droplet actuator that includes a magnetic shield for reducing the crossover of magnetic fields;

FIG. 7 illustrates a top view of a section of a droplet actuator that includes a magnet having poles that are oppositely arranged for reducing the crossover of magnetic fields;

FIG. 8 illustrates a map of magnetic fields that are arranged in a relationship such as the one shown in FIG. 7;

FIG. 9 illustrates a top view of a section of a droplet actuator arranged to reduce carryover at a droplet detection region;

FIGS. 10A and 10B illustrate a top view of a section of a droplet actuator configured to reduce carryover;

FIGS. 11A, 11B, and 11C illustrate side views of section of a droplet actuator configured for improving the sensitivity of droplet detection;

FIG. 12 illustrates a top view of a section of a droplet actuator configured for improving the sensitivity of droplet detection;

FIG. 13 illustrates a side view of a section of a droplet actuator configured for improving the sensitivity of droplet detection;

FIGS. 14A and 14B illustrate top views of a modular droplet actuator assembly, which provides a universal assembly for orienting a magnet assembly to a droplet actuator; and

FIG. 15 illustrates a side view of a modular droplet actuator assembly, which is another nonlimiting example of a universal assembly for orienting a magnet assembly to a droplet actuator.

8 DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, among other things, to droplet actuators configured for immobilizing magnetically-responsive beads and to methods of making and using such droplet actuators. As an example, the droplet actuators are useful for immobilizing beads in droplets on the droplet actuators, thereby facilitating the execution of protocols which require immobilization of such beads, such as bead washing protocols. The invention also provides techniques for reducing or eliminating carryover of substances from droplet to droplet in a droplet actuator and techniques for maximizing signal detection on a droplet actuator.

8.1 Bead Loss During Droplet Splitting

FIGS. 1A and 1B illustrate first and second top views, respectively, of a droplet actuator 100 in use during a first and second phase, respectively, of a droplet splitting operation. Droplet actuator 100 makes use of an arrangement that is not suitably arranged for efficiently splitting a droplet using the specific technique shown without loss of magnetically responsive beads. Droplet actuator 100 includes a first substrate 110 and a second substrate (not shown) arranged with a gap therebetween, which serves as a fluid path. First substrate 110 includes a set of droplet operation electrodes 112 configured for conducting droplet operations on slug-shaped droplet 114, which is suspended in a filler fluid and includes magnetically responsive beads 116. A magnet 118 may be arranged in sufficient proximity to droplet operation electrodes 112 to permit some degree of immobilization of the magnetically responsive beads 116.

FIGS. 1A and 1B show a splitting operation that is taking place in the presence of a magnetic field produced within droplet actuator 100 by magnet 118. The placement of magnet 118 is not suitable for localizing substantially all of magnetically responsive beads 116 in a centralized location (e.g., away from the edges of the droplet) within the portion of droplet 114 that is selected to retain the beads after the splitting operation. Consequently, a certain quantity of magnetically responsive beads 116 bridge splitting zone 120 during the droplet splitting operation, as shown in FIG. 1A, and a loss of beads results, as shown in FIG. 1B. FIG. 1B shows a first droplet 122 that contains a certain quantity of the original quantity of magnetically responsive beads 116 and a second droplet 124 that contains a certain remaining quantity of the original quantity of magnetically responsive beads 116. In other words, the end result of the illustrated splitting operation is a loss of magnetically responsive beads 116. The inventors have discovered that a contributing factor to the loss of beads is that a full quantity of magnetically responsive beads 116 is not suitably attracted, immobilized, and retained at a centralized location within droplet 114 and/or at a sufficient distance from splitting zone 120.

8.2 Magnet Configurations for Preventing/Reducing Bead Loss

Among other things, the invention provides improved droplet actuators that have various magnet configurations in which magnets are arranged for efficiently splitting bead-containing droplets and washing magnetically responsive beads are described with reference to FIGS. 2, 3, 4, 5A, and 5B. These figures illustrate nonlimiting examples of magnet configurations in combination with a droplet actuator splitting droplets with little or no bead loss, and are, among other things, useful for efficiently washing magnetically responsive beads. One or more magnets may be arranged in proximity to a droplet on a droplet actuator such that magnetically responsive beads are suitably attracted and immobilized within the droplet, preferably at a centralized location away from the neck that forms during splitting operations. In this approach, all or substantially all magnetically responsive beads are retained within a single droplet upon completion of a droplet splitting operation. Similarly, the spitting operation may be performed in a droplet slug which is at a distance from the immobilized beads which is sufficient to reduce or eliminate bead loss. As will be explained with respect to the examples below, one or more magnets may be arranged with respect to the droplet actuator structure above, below, and/or beside, the magnetically-responsive bead-containing droplet and any combinations thereof to achieve this purpose.

8.2.1 Position of Magnet Relative to Splitting Zone

FIGS. 2A and 2B illustrate side and top views of a droplet actuator 200. In this embodiment, the magnet is placed at a position which is sufficiently distant from the portion of the droplet which is breaking during the splitting operation, splitting zone 224 (or vice versa, the splitting zone may be said to be placed at sufficient distance from the magnet position), to reduce or eliminate bead loss during a droplet splitting operation. Further, the magnet is positioned so that the beads are generally centrally located along a lateral diameter L of the droplet (top view). Droplet actuator 200 includes a first substrate 210 and a second substrate 212 separated to provide a gap for conducting droplet operations, though only one substrate is required. A set of droplet operation electrodes 214 is associated with one or both of the substrates and arranged for conducting one or more droplet operations. Droplet actuator 200 may include a magnet 216 that is arranged in sufficient proximity to droplet 218/222 to substantially immobilize magnetically responsive beads 220 during a droplet splitting operation. For example, the magnet may be arranged as a component of the droplet actuator and/or in sufficient proximity to the droplet actuator to immobilize the magnetically responsive beads in droplet 218/222 in the gap. Droplet 218/222 may be surrounded by a filler fluid (not shown). Droplet 218/222 contains a quantity of magnetically responsive beads 220 immobilized by magnet 216.

Magnet 216 is positioned relative to one or more droplet operation electrodes 214, in order to localize beads 220 in a region of the portion of the droplet 218/222 that is to form droplet 218 without permitting substantial loss of beads 218 during the droplet splitting operation to droplet 222.

In operation, a splitting operation is achieved without substantial loss of magnetic beads 220 by: as shown in 201, providing a droplet actuator 200 with electrodes activated (ON) to form combined droplet 218/222 and magnet 216 is arranged in a position which causes substantially all of magnetically responsive beads 220 to be attracted to magnet 216 in a zone of droplet 218/222 that prevents substantial loss of magnetically responsive beads 220 to droplet 222. Magnet 216 may be arranged so that magnetically responsive beads 220 attracted thereto are localized at a generally centralized location along lateral diameter L within combined droplet 218/222 and away from the droplet split zone 224. During a droplet splitting operation as shown in 202, an intermediate electrode is deactivated (OFF) to cause splitting at split zone 224. Substantially all magnetically responsive beads 220 are retained in droplet 218, and droplet 222 is formed and is substantially free of magnetically responsive beads 220, as shown in 203.

A process for washing magnetically responsive beads 220 may, in one embodiment, involve the repetition of droplet merging (with a wash droplet), bead immobilization, splitting, and bead resuspension operations until acceptable levels of washing are achieved.

8.2.2 Two-Magnet Arrangement to Produce Bead Column

FIG. 3 illustrates a side view of a droplet actuator 300. Droplet actuator 300 is generally configured as described in FIG. 2, except that it includes two magnets, magnet 310 a and 310 b, arranged below and above droplet 218. The magnets may be integral with droplet actuator 300 and/or arranged in close proximity to the outer side of first substrate 210 and second substrate 212. In general, the magnets 310 a and 310 b may be arranged such that opposite poles are facing one another. In one example, the north or positive pole of magnet 310 a is facing the south or negative pole of magnet 310 b, as shown in FIG. 3.

Magnets 310 a and 310 b may be separate magnets or, alternatively, magnets 310 a and 310 b may be opposite poles of a single U-shaped, C-shaped, or horseshoe-shaped permanent magnet or electromagnet. The arrangement of magnets 310 a and 310 b may cause magnetically responsive beads 220 to be immobilized and retained in a column-shaped cluster. The magnets are preferably arranged to localize beads within droplet 218 in a position which is away from splitting zone 224 in the portion of the combined droplet (not shown) in which the beads are to be retained. Further, the magnets are preferably aligned to centrally localize the beads along lateral diameter L within the combined droplet (not shown).

8.2.3 Multiple Magnet Pairs to Centralize Beads

FIGS. 4A and 4B illustrate side views of a droplet actuator 400. Droplet actuator 400 is generally configured like droplet actuator 200 described in FIG. 2, except that it includes four magnets arranged at positions surrounding the droplet. In general, the arrangement illustrates the embodiment in which multiple magnet pairs with positive/negative poles facing each other are arranged to generally centrally locate beads 220 in the combined droplet prior to splitting. As illustrated in FIG. 4B, the beads are generally centrally located along a vertical dimension V and a lateral dimension L. In the example illustrated, droplet actuator 400 includes four magnets, such as a magnet 410 a, 410 b, 410 c, and 410 d.

Magnets 410 a and 410 b may be arranged in close proximity to the droplet, equally spaced on either side of the droplet, with opposite poles facing each other. For example, the north pole of magnet 410 a may face the south pole of magnet 410 b. Magnets 410 c and 410 d may be arranged in close proximity to the droplet, equally spaced on either side of the droplet, with opposite poles facing each other. For example, the north pole of magnet 410 d may face the south pole of magnet 410 c. Magnet pair 410 a/410 b may generally be aligned at right angles around the droplet relative to magnet pair 410 c/410 d. In the illustrated embodiment, magnet pair 410 a/410 b has a vertical orientation around the droplet, and magnet pair 410 c/410 d has a horizontal orientation around the droplet. Any orientation around the droplet achieving the generally central localization of beads along lateral dimension L and vertical dimension V will suffice to achieve the desired central immobilization.

Magnets 410 a and 410 b may be arranged in close proximity to the outer side of first substrate 210 and second substrate 212, respectively, such that the magnetic field of magnets 410 a and 410 b may pass through the gap between first substrate 210 and second substrate 212 of droplet actuator 400. Magnets 410 a and 410 b are arranged such that opposite poles are facing one another. In one example, the north pole of magnet 410 a is facing the south pole of magnet 410 b, as shown in FIG. 4. Similarly, magnets 410 c and 410 d are arranged in close proximity to a first side and a second side, respectively, of droplet actuator 400, such that the magnetic field of magnets 410 c and 410 d may pass through the gap of droplet actuator 400 and perpendicular to the magnetic field of magnets 410 a and 410 b. Magnets 410 c and 410 d are arranged such that opposite poles are facing one another. In one example, the north pole of magnet 410 d is facing the south pole of magnet 410 c, as shown in FIG. 4.

Magnets 410 a and 410 b may be separate magnets or, alternatively, magnets 410 a and 410 b may be opposite poles of a single U-shaped, C-shaped, or horseshoe-shaped permanent magnet or electromagnet. Similarly, magnets 410 c and 410 d may be separate magnets or, alternatively, magnets 410 c and 410 d may be opposite poles of a single U-shaped, C-shaped, or horseshoe-shaped permanent magnet or electromagnet. Because the magnetic field of magnets 410 a and 410 b and magnets 410 c and 410 d, respectively, intersect at the center of the fluid path within droplet actuator 400, the magnetically responsive beads 220 are magnetically immobilized and retained in a cluster that is centralized within the combined droplet and retained in droplet 218 following the splitting operation.

8.2.4 Illustration of Splitting with Substantially No Bead Loss

FIGS. 5A and 5B illustrate a first and second top view, respectively, of a droplet actuator 500 during a first and second phase, respectively, of a droplet splitting operation. Droplet actuator 500 may alternatively be configured like any of the example droplet actuators 200, 300, and 400. Droplet actuator 500 of FIGS. 5A and 5B makes use of magnet forces that are suitably arranged for use in a splitting operation designed to result in substantially complete retention of beads in a single droplet, such as a process for washing magnetically responsive beads.

In particular, FIGS. 5A and 5B show a splitting operation that is taking place at a sufficient distance from localized beads 220 to permit a splitting operation that results in substantially complete retention of magnetically responsive beads in droplet 218 and a droplet that is substantially free of magnetically responsive beads. The position of magnet face 510 is suitably arranged to magnetically immobilize substantially all of magnetically responsive beads 220 at a centralized location within the droplet and at a distance from the splitting zone that is sufficient to achieve the desired retention of magnetically responsive beads 220 in droplet 218. As a result, substantially no quantity of magnetically responsive beads 220 bridges a splitting zone 512 during the droplet splitting operation, as shown in FIG. 5A, and substantially no loss of beads occurs. FIG. 5B shows droplet 218 that contains substantially all magnetically responsive beads 220. In other words, the end result of a splitting operation that takes place substantially outside of the magnet forces is that there is substantially no loss of magnetically responsive beads 220 because substantially all of magnetically responsive beads 220 are suitably attracted, immobilized, and retained at a centralized location within the fluid.

8.3 Droplet Actuator Configurations with Magnets 8.3.1 Droplet Actuator with Magnetic Shield

FIG. 6 illustrates a side view of a droplet actuator 600 that includes a magnetic shield for reducing the crossover of magnetic fields. Droplet actuator 600 includes a top plate 610 and a bottom plate 614 that are arranged having a gap 618 therebetween. An arrangement of electrodes 622, e.g., electrowetting electrodes, may be associated with bottom plate 614 for performing droplet operations and an electrode, such as reservoir electrode 626 that is associated with a fluid reservoir 630 that contains a quantity of fluid 634. One or more droplets (not shown) may be dispensed from the quantity of fluid 634 of reservoir 630 for manipulation along electrodes 622. Additionally, fluid 634 and any droplets dispensed therefrom may optionally contain beads (not shown), which may, in some cases, be magnetically responsive.

Droplet actuator 600 further includes a magnet 638 that is arranged in proximity to the one or more electrodes 622. Magnet 638 may be arranged in sufficient proximity to electrodes 622 in order to permit immobilization of magnetically responsive beads (not shown), e.g., in a droplet positioned on an electrode. In one example, the purpose of magnet 638 is to magnetically immobilize and retain magnetically responsive beads during a droplet splitting operation, e.g., a splitting operation that may be performed in a process for washing magnetically responsive beads.

Additionally, droplet actuator 600 includes a magnetic shield 642 that is arranged in sufficient proximity to fluid reservoir 630 to shield the contents thereof from nearby magnetic fields, such as the magnetic field of magnet 638. Magnetic shield 642 may, for example, be formed of Mu-metal that has sufficiently high magnetic permeability and that is suitable to reduce, preferably substantially eliminate, unwanted magnetic fields from the magnet within fluid reservoir 630. In one example, magnetic shield 642 may be formed of Mu-metal that is supplied by McMaster-Carr (Elmhurst, Ill.). Other examples of magnetic shield 642 materials include Permalloy, iron, steel and nickel.

Droplet actuator 600 is not limited to one magnetic shield and one magnet only, any number of magnetic shields and magnets may be installed therein. Therefore, by use of one or more magnetic shields, the exposure of magnetic beads (not shown) within droplets to magnetic fields may be limited to desired regions only of droplet actuator 600. Magnetic shields may be included on any surface of the droplet actuator and in any arrangement which facilitates suitable shielding.

In one example application, a droplet actuator may be employed to perform multiple assays in parallel and, consequently, there may be a need for generally simultaneous washing of the various magnetic beads that may be manipulated within multiple lanes of electrodes. Without magnetic shields, a wash operation or assay that is performed at a certain location using an associated magnet may be affected by the magnetic field of a distant magnet (i.e., crossover of magnetic fields). By contrast, crossover of magnetic fields between any two magnets may be reduced, preferably substantially eliminated, via the strategic placement of one or more magnetic shields, such as magnetic shield 642, within the droplet actuator.

8.3.2 Droplet Actuator with Alternating Magnet Configuration

FIG. 7 illustrates a top view of a droplet actuator 700 that includes a magnet whose poles are oppositely arranged for reducing the crossover of magnetic fields. Droplet actuator 700 includes an arrangement of electrodes 710, e.g., electrowetting electrodes, for performing droplet operations on one or more droplets (not shown). Additionally, a magnet 714 is arranged in close proximity to a first lane of electrodes 710, a magnet 718 is arranged in close proximity to a second lane of electrodes 710, a magnet 722 is arranged in close proximity to a third lane of electrodes 710, and a magnet 726 is arranged in close proximity to a fourth lane of electrodes 710. Magnets 714, 718, 722, and 726 may be arranged in sufficient proximity to electrodes 710 in order to permit immobilization of magnetically responsive beads (not shown) within one or more droplets (not shown) located on one or more of the electrodes.

In order to reduce, preferably substantially eliminate, crossover of magnetic fields between adjacent magnets, the poles of adjacent magnets are oppositely arranged, which causes the adjacent magnetic fields to cancel. For example and referring again to FIG. 7, the north pole of magnet 722 is oriented upward, and the south pole of magnet 726 is oriented upward. In this way, the magnetic fields are cancelled and the crossover of magnetic fields between magnets 714, 718, 722, and 726 may be reduced, preferably substantially eliminated.

FIG. 8 illustrates a map 800 of magnetic fields arranged in a relationship such as the one shown in FIG. 7.

8.4 Other Techniques

The invention also provides techniques for reducing carryover in a droplet actuator, as well as techniques for improving detection operations.

8.4.1 Technique for Reducing Carryover in a Droplet Actuator

FIG. 9 illustrates a top view of a droplet actuator 900 by which an operation to reduce carryover at a droplet detection region may be performed. Droplet actuator 900 includes an arrangement of electrodes 910, e.g., electrowetting electrodes, for subjecting droplets to droplet operations. Additionally, droplet actuator 900 includes a designated detection region 914 at, for example, a certain electrode 910. Detection region 914 is used to detect droplets positioned thereon or passing therethrough during droplet operations. In one example, droplet detection is performed using a photomultiplier tube (PMT) or photon-counting PMT that is associated with detection region 914. A PMT (not shown) is used to measure light (i.e., detect photons) emitted from a droplet (e.g., due to fluorescence and/or chemiluminescence) when at the electrode that is associated with detection region 914.

In some cases, a build up of substances at a detection region, such as detection region 914, may occur due to carryover, which involves beads or other substances being left behind on surfaces and/or in filler fluid during droplet operations. Carryover may interfere with accurate detection of signals from subsequent droplets and/or interfere with droplet operations by affected electrodes.

Referring again to FIG. 9, a droplet sequencing operation of the invention reduces, preferably substantially eliminates, carryover at detection region 914 by providing a series of alternating assay droplets 918 and wash droplets 922. In one example, an assay droplet 918 a passes through detection region 914, followed by a wash droplet 922 a, followed by an assay droplet 918 b, followed by a wash droplet 922 b, followed by an assay droplet 918 c, followed by a wash droplet 922 c, followed by an assay droplet 918 d, which is followed by a wash droplet 922 d. As assay droplets 918 a, 918 b, 918 c, and 918 d have the potential to degrade the function of detection region 914 due to crossover, wash droplets 922 a, 922 b, 922 c, and 922 d perform a cleaning operation of the surfaces that are associated with detection region 914. The cleaning process of the invention is not limited to the sequence that is shown in FIG. 9. Any sequence is possible as long as the sequence includes a suitable number of wash droplets in order to suitably clean the detection region. For example, depending on the requirements of the specific assay, multiple wash droplets may be provided between assay droplets and/or multiple assay droplets may be provided between wash droplets, e.g., AAWAAWAAW, AAAWAAAWAAAW, AWWAWWAWW, AWWWAWWWAWWW, AAWWAAWWAAWW, AAAWWWAAAWWWAAAWWW, etc., where A=assay droplet and W=wash droplet. It should be noted that it is not necessary for assay and wash droplets to be the same size. Assay droplets may be larger or wash droplets may be larger. The larger droplet may be subjected to droplet operations as a slug (e.g., a slug occupying 4 electrodes) or as a single large droplet (e.g., a 4× droplet occupying as many electrodes as it naturally covers without being formed into a slug). Each arrangement may result in a different cleaning result. It is also not necessary for the assay droplets and wash droplets to follow the same path. For example, their paths may intersect at the location needing to be cleaned.

FIGS. 10A and 10B illustrate a top view of a droplet actuator 1000 by which another operation for reducing carryover may be performed. Droplet actuator 1000 includes an arrangement of electrodes 1010, e.g., electrowetting electrodes, for performing droplet operations on one or more droplets, such as assay droplets 1014 and 1018 (FIG. 10A) and wash droplet 1022 (FIG. 10B). Additionally, a magnet 1026 is arranged in close proximity to a certain electrode 1010. Magnet 1026 may be arranged in sufficient proximity to the certain electrode 1010 in order to permit immobilization of magnetically responsive beads within one or more droplets, such as magnetic beads 1030 within assay droplet 1014.

During, for example, a droplet split operation, a quantity of “satellite” droplets may be left behind at the point at which the split occurs. For example and referring to FIG. 10A, a droplet split operation by which assay droplet 1014 is formed by splitting from assay droplet 1018 may result in a certain quantity of satellite droplets 1034 being left behind upon a certain electrode 1010. Satellite droplets, such as satellite droplets 1034, may be the source of carryover (cross contamination) from one droplet to another, which is not desired. FIG. 10B illustrates that a wash droplet, such as wash droplet 1022, may be transported along electrodes 1010 subsequent to the assay operations of, for example, assay droplets 1018 and 1014 in order to capture satellite droplets 1034 and transport them away prior the next assay operation occurs. In this way, electrodes 1010 are cleaned between assay operations. The cleaning process of the invention is not limited to the sequence that is shown in FIGS. 10A and 10B. Any sequence is possible as long the sequence includes a suitable number of wash droplets in order to suitably clean the electrodes.

8.4.2 Improving Detection Operations in a Droplet Actuator

FIGS. 11A, 11B, and 11C illustrate side views of droplet actuator 1100 by which respective operations for improving the sensitivity of droplet detection may be performed. Droplet actuator 1100 includes a top plate 1110 and a bottom plate 1114 that are arranged having a gap 1118 therebetween. An arrangement of electrodes 1122, e.g., electrowetting electrodes, may be associated with bottom plate 1114 for performing droplet operations on a droplet 1126. A PMT window 1130 may be associated with top plate 1110, by which a PMT (not shown) is used to measure light (i.e., detect photons 1134) emitted from droplet 1126.

FIG. 11A shows a method of improving the sensitivity of droplet detection by spreading out a droplet, such as droplet 1126, in order to increase the surface area that is exposed to PMT window 1130 and, thus, increase the number of photons 1134 that may be detected. A droplet may be spread linearly across one or more electrodes 1122 depending on the volume of the droplet. In the case of a small-volume droplet, a buffer droplet may be added to make the droplet larger, as long as losses due to dilution by the buffer droplet are offset by the increased droplet area exposed to the PMT. For example, FIG. 11B shows droplet 1126 that is spread continuously across multiple electrodes 1122, which increases the number of photons 1134 that may reach PMT window 1130 and may be detected by the PMT.

FIG. 11C shows a scenario wherein droplet 1126 is split up into multiple droplets 1126, such as droplets 1126 a, 1126 b, 1126 c, and 1126 d that are on multiple electrodes 1122. Again, the surface area that is exposed to PMT window 1130 is increased, which increases the number of photons 1134 that may reach PMT window 1130 and may be detected by the PMT. Alternatively and referring again to FIGS. 11A, 11B, 11C, the spreading of a droplet, such as droplet 1126, is not limited to linear spreading only. The droplet may be spread two dimensions, such as across a grid or array or electrodes 1122, in order to increase the surface area that is exposed to PMT window 1130. Alternatively, one or more large-area electrodes may be provided, across which one or more droplets may be spread.

FIG. 12 illustrates a top view of a droplet actuator 1200 for improving the sensitivity of droplet detection. Droplet actuator 1200 includes an arrangement of electrodes 1210, e.g., electrowetting electrodes, for performing droplet operations on multiple droplets 1214. Additionally, droplet actuator 1200 may include a droplet detection region 1218 that has an associated PMT (not shown) for measuring light that is emitted from a certain droplet 1214 when present. In order to reduce, preferably substantially eliminate, the carryover of light from a distant droplet 1214 to droplet detection region 1218, a minimum distance d is maintained in all directions between the outer perimeter of droplet detection region 1218 and any distant droplets 1214 within droplet actuator 1200, as shown in FIG. 12. The minimum distance d is sufficiently large to reduce, preferably substantially eliminate, the carryover of light from a distant droplet 1214 to droplet detection region 1218. As a result, in a droplet actuator that includes multiple droplets 1214, a spacing is maintained during detection between the target droplet 1214 that is being measured and a distant droplet 1214, such that the carryover of light from the distant droplet 1214 to droplet detection region 1218 is reduced, preferably substantially eliminated. As a specific case the distance d is an integer multiple m of a unit electrode size, and the droplet detection may be conducted on a set of electrodes which are electrically connected as an m phased bus. Alternatively, FIG. 13 (described below) describes a scenario wherein the real-estate with a droplet actuator is limited and, therefore, sufficient spacing between droplets, as described in FIG. 12, may not be achieved.

In an alternative embodiment, cross-over from nearby droplets is eliminated by using optical elements, such as one or more lenses, which focus only light from the droplet being interrogated onto the sensor and eliminates signal from other droplets.

FIG. 13 illustrates a side view of a droplet actuator 1300 for improving the sensitivity of droplet detection. Droplet actuator 1300 includes a top plate 1310 and a bottom plate 1314 that are arranged having a gap 1318 therebetween. An arrangement of electrodes 1322, e.g., electrowetting electrodes, may be associated with bottom plate 1314 for performing droplet operations on droplets, such as a droplet 1326 and a droplet 1330. A PMT window 1334 may be associated with top plate 1310, by which a PMT (not shown) is used to measure light (i.e., detect photons 1338) that is emitted from, for example, droplet 1326. Because the spacing between, for example, droplet 1326 at PMT window 1334 and distant droplet 1330 is not suitably sufficient to avoid the carryover of light from droplet 1330 to PMT window 1334, a mask 1342 is provided upon top plate 1310. The purpose of mask 1342 is to block light from a distant droplet from carrying over to PMT window 1334, which is the detection region of a target droplet.

Mask 1342 may be formed upon top plate 1310 via a layer of any light-absorbing material, as long as the material that is used is compatible with the electrowetting process and does not unduly interfere with the droplet actuator operations. In one example, mask 1342 may be formed by applying a layer of black paint to top plate 1310, such that one or more windows, such as PMT window 1334, are provided in selected detection regions of droplet actuator 1300. In the example shown in FIG. 13, mask 1342 reduces, preferably substantially eliminates, the carry over of light from distant droplet 1330 to the target droplet 1326 at PMT window 1334. In another example the mask 1342 is formed by an opaque conductor on the side of the top plate 1310 facing the droplet. The conductor may, for example, be aluminum, chromium, copper, or platinum. The conductor may additionally serve as an electrical reference electrode.

8.5 Droplet Actuators with Magnet Assemblies

FIGS. 14A and 14B illustrate top views of a modular droplet actuator assembly 1400, which is a nonlimiting example of a universal assembly for orienting a magnet assembly to a droplet actuator. Modular droplet actuator assembly 1400 may include, for example, a mount 1410, a magnet assembly 1420, and a droplet actuator 1430. FIG. 14A shows modular droplet actuator assembly 1400 when disassembled. FIG. 14B shows modular droplet actuator assembly 1400 when assembled.

Magnet assembly 1420 may include a substrate 1424 upon which is mounted one or more magnets 1428, as shown in FIG. 14A. The magnets 1428 may be permanently affixed to substrate 1424 or may be removable. Removable magnets 1428 facilitate selection by a user of magnets having desired properties, such as desired magnet strength. In one embodiment, a droplet actuator instrument is provided with a droplet actuator assembly 1400 including a mount 1410 and a magnet assembly 1420 without magnets. In another embodiment, the user is also provided with magnets having specified properties which may be affixed by the user to the magnet assembly 1420. In another embodiment, the user is also provided with sets of magnets having various specified characteristics such that the user may select a set of one or more magnets having desired properties and affix the selected set to the magnet assembly 1420.

Magnets may be marked or coded (e.g., color coded) to facilitate selection of magnets having appropriate properties, as well as marked to show the orientation of the magnet's magnetic field (e.g., by color coding or otherwise marking the North and South faces of the magnets). Similarly, the magnet assembly 1420 may be marked to show the desired orientation of magnets inserted therein, and in some embodiments, magnets may be shaped to permit them to be affixed to the magnet assembly 1420 only in an appropriate orientation.

Moreover, in another embodiment, the user may be provided with magnet assemblies 1420 having magnets already affixed thereto, wherein the magnet assemblies 1420 each have different magnet configurations, e.g., sets of magnets having different properties. The user may select the magnet configuration having magnets having properties appropriate to the user's desired use for the instrument. Magnet assembly 1420 may be marked or otherwise color coded to facilitate selection by the user. Magnet properties may, for example, be selected based on the properties of beads selected by the user.

Droplet actuator 1430 may include a substrate 1434 upon which is an arrangement of electrodes 1438, e.g., electrowetting electrodes, as shown in FIG. 14A. A second (top) substrate may also be included (not shown).

Magnet assembly 1420 is designed such that magnets 1428 substantially align with certain electrodes 1438 of interest on droplet actuator 1430. For example, in some embodiments, parallel configurations of magnets may be present for conducting parallel assay steps on droplet actuator 1430. Magnets may be configured and oriented, for example, according to the various configurations and orientations described herein.

Mount 1410 may serve as a universal platform for mounting a magnet assembly, such as magnet assembly 1420, and a droplet actuator, such as droplet actuator 1430. In one embodiment, mount 1410 is configured to accept a wide variety of magnet assemblies 1420 and a wide variety of droplet actuators 1430. Magnet assembly 1420 may include one or more magnets arranged in any of a variety of patterns and employing any of a variety of magnet properties. FIG. 14 illustrates a row of magnets, but magnets may also be provided in a grid or any arrangement that places the magnets in their proper position in relation to the droplet actuator 1430 in order to facilitate desired operations on the droplet actuator.

In one example, magnet assembly 1420 and droplet actuator 1430 may be installed into mount 1410 via respective fittings 1418 and 1414. Fittings may, for example, involve slots into which the mount 1410 may be fitted, openings on the mount 1410 for accepting posts on the magnet assemblies 1420 or vice versa, openings on the mount 1410 for accepting screws on the magnet assemblies 1420, threaded posts for accepting bolts, various spring loaded mechanisms, recessed trays, complimentary fittings, and the like. Any mechanism which facilitates sufficiently secure attachment to permit the device to function for its intended purpose will suffice.

Further, the mount 1410 may include multiple fittings for multiple possible positions of magnet assembly 1420 and/or droplet actuator 1430, and/or mounting of multiple magnet assemblies 1420 and/or multiple droplet actuators 1430 in a single mount 1410. Further, mount 1410 may be configured to permit magnet assemblies 1420 to be mounted above, below and/or beside the droplet actuator 1430, i.e., in any relationship with the droplet actuator. With droplet actuator 1430 installed in modular mount 1410, any magnet assembly of interest, such as magnet assembly 1420, may be inserted into modular droplet actuator assembly 1400 via, for example, the slot.

FIG. 15 illustrates a side view of a modular droplet actuator assembly 1500, which is another nonlimiting example of a universal assembly for orienting a magnet assembly to a droplet actuator, similar to the assembly 1400 shown in FIG. 14, except that mount 1410 is replaced with a mount 1510. Mount 1510 includes recessed trays into which magnet assembly 1420 and droplet actuator 1430 may be fitted in order to provide a “drop in” method of loading.

Referring to FIGS. 14A, 14B, and 15, an aspect of the invention is that the slots or other attachment means serve to orient the magnet substrate and the droplet actuator so as to align the magnets on the magnet assembly with the appropriate electrodes or electrode paths on the droplet actuator. In this manner, the magnets may be removed when not needed for an assay. Additionally, different magnet mounts with different distributions of magnets may be provided for different types of assays or different droplet actuator layouts.

8.6 Magnets

In addition to other aspects already described, it should be noted that magnets selected for use with the invention may be permanent or electromagnets. There may be a relationship between the magnetically responsive content of the beads in the droplet and the magnetic strength/pull force. Therefore, the magnetic strength/pull force of the magnet may be selected relative to the responsiveness of the magnetic beads such that it is:

-   -   sufficiently strong relative to the magnetic responsiveness of         the beads to substantially immobilize magnetically responsive         beads;     -   not so strong relative to the magnetic responsiveness of the         beads that it significantly magnetizes the beads and, thus,         causes irreversible formation of clumps of beads;     -   not so strong relative to the magnetic responsiveness of the         beads that resuspension occurs poorly when the magnet field is         removed; and/or     -   not so strong relative to the magnetic responsiveness of the         beads that the beads are pulled out of the droplet altogether.

In some embodiments, the magnet may have high magnetic strength (in Tesla) with lesser pull force (in pounds). In one example, magnet is a neodymium permanent magnet that has a surface field strength of about 1 Tesla (T). In another example, the magnet is an electromagnet that has a surface field strength of about 1 T, which may be turned on and off electronically. Where a permanent magnet is used, the magnet may be moved away from the magnetically responsive bead-containing droplet for uses in which it is desirable to remove the influence of the magnetic field. While not limited to the following ranges, it is understood that ranges of magnetic strength that generally encompasses the useful strength of the present invention can include: a broad range of 0.01 T to 100 T (pulsed) or 45 T (continuous); an intermediate range of 0.01 T to 10 T; and a narrow range of 0.1 T to 1 T (preferably 0.5 T).

8.7 Droplet Composition

Droplets including magnetic beads and subjected to droplet splitting operations may include any of a wide variety of samples, reagents, and buffers useful for conducting assays using the beads. For example, during washing, the droplet may include a buffer, such as a phosphate-buffered saline (PBS) buffer with a surfactant that is suitable for use in magnetic based immunoassays. Preferred surfactants are those which facilitate immobilization and/or resuspension of beads following immobilization by magnetic forces. The surfactant and amount of surfactant may be adjusted to provide a substantial improvement in resuspension as compared to a control solution lacking the surfactant. In one embodiment, the droplet includes PBS buffer with about 0.01% Tween® 20.

A hydrophilic polymer and/or surfactant may be included in the droplet to facilitate retention and resuspension of magnetically responsive beads during splitting operation. The droplet may include a wide variety of liquids immiscible with the filler fluid. Examples of buffers include, but are not limited to, phosphate-buffered saline (PBS) buffer and Tris buffer saline. In one embodiment, the droplet fluid includes a buffer, such as the PBS buffer, and any surfactant that is suitable for use in magnetic based immunoassays.

Preferred hydrophilic polymers and surfactants are those which facilitate resuspension of beads following immobilization by magnetic forces. The surfactant and amount of surfactant may be adjusted to provide a substantial improvement in resuspension as compared to a control solution lacking the surfactant. Examples of surfactants that are suitable for use in magnetic based immunoassays include, but are not limited to, polysorbate 20, which is commercially known as Tween® 20, and Triton X-100. Tween® 20 may be supplied by, for example, Pierce Biotechnology, Inc. (Woburn, Mass.). Triton® X-100 may be supplied by, for example, Rohm & Haas Co (Philadelphia, Pa.). In one example, the droplet fluid within the droplet actuator is a mix of PBS with Tween® 20 in the range of from about 0.001% to about 0.1%. In another example, the droplet fluid within the droplet actuator is a mix of PBS with about 0.01% Tween® 20.

Other examples include pluronic surfactants, polyethylene glycol (PEG), methoxypolyethylene glycol (MPEG), poly-sorbate(polyoxyethylene sorbitan monooleates or Tween®), polyoxyethylene octyl phenyl ether (Triton X-1000), polyvinyl pyrollidone, polyacrylic acid (and crosslinked polyacrylic acid such as carbomer), polyglycosides (nonionic glycosidic surfactants such as octyl glucopyranoside) and soluble polysaccharides (and derivatives thereof) such as heparin, dextrans, methyl cellulose, propyl methyl cellulose (and other cellulose esters and ethers), dextrins, maltodextrins, galactomannans, arabinogalactans, beta glucans, alginates, agar, carrageenan, and plant gums such as xanthan gum, psyllium, guar gum, gum traganth, gum karya, gum ghatti and gum acacia. The particular additive can be selected for maximum compatibility with a specific microfluidic sample.

8.8 Droplet Actuator

For examples of droplet actuator architectures that are suitable for use with the present invention, see U.S. Pat. No. 6,911,132, entitled, “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on Jun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No. 11/343,284, entitled, “Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board,” filed on filed on Jan. 30, 2006; U.S. Pat. No. 6,773,566, entitled, “Electrostatic Actuators for Microfluidics and Methods for Using Same,” issued on Aug. 10, 2004 and U.S. Pat. No. 6,565,727, entitled, “Actuators for Microfluidics Without Moving Parts,” issued on Jan. 24, 2000, both to Shenderov et al.; and International Patent Application No. PCT/US 06/47486 to Pollack et al., entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006, the disclosures of which are incorporated herein by reference. Droplet actuator techniques for immobilizing magnetic beads and/or non-magnetic beads are described in the foregoing international patent applications and in Sista, et al., U.S. Patent Application No. 60/900,653, filed on Feb. 9, 2007, entitled “Immobilization of magnetically-responsive beads during droplet operations”; Sista et al., U.S. Patent Application No. 60/969,736, filed on Sep. 4, 2007, entitled “Droplet Actuator Assay Improvements”; and Allen et al., U.S. Patent Application No. 60/957,717, filed on Aug. 24, 2007, entitled “Bead washing using physical barriers,” the entire disclosures of which is incorporated herein by reference. Combinations of these various techniques are within the scope of this invention.

8.9 Fluids

For examples of fluids that may be subjected to droplet operations of the invention, see the patents listed in section 8.8, especially International Patent Application No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In some embodiments, the droplet is a sample fluid, such as a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes. In some embodiments, the fluid that is loaded includes a reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and/or buffers. In some embodiments, the fluid that is loaded includes a reagent, such as a reagent for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids.

8.10 Filler Fluids

As noted, the gap is typically filled with a filler fluid. The filler fluid may, for example, be a low-viscosity oil, such as silicone oil. Other examples of filler fluids are provided in International Patent Application No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006.

8.11 Washing Magnetically Responsive Beads

For protocols making use of beads, droplets with beads can be combined using droplet operations with one or more wash droplets. Then, while retaining the beads (e.g., physically or magnetically) using magnet configurations of the invention, the merged droplet may be divided using droplet operations into two or more droplets: one or more droplets with beads and one or more droplets without a substantial amount of beads. In one embodiment, the merged droplet is divided using droplet operations into one droplet with beads and one droplet without a substantial amount of beads.

Generally, each execution of a washing protocol results in retention of sufficient beads for conducting the intended assay without unduly detrimental effects on the results of the assay. In certain embodiments, each division of the merged droplet results in retention of more than 90, 95, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99, 99.9. 99.99, 99.999, 99.9999, 99.99999, or 99.999999 percent of beads. In other embodiments, each execution of a washing protocol to achieve a predetermined reduction in the concentration and/or amount of removed substance results in retention of more than 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99, 99.9. 99.99, 99.999, 99.9999, 99.99999, or 99.999999 percent of beads. In still other embodiments, the amount of retained beads is calculated and the results are adjusted accordingly.

In some embodiments, beads can be washed in reservoirs in which the bead-containing droplet and wash droplets are combined, beads are retained (for example by a magnet, by physical structures, electrostatic forces), and droplets lacking beads are dispensed from the reservoir using droplet operations. For example, beads can be washed by dilute-and-dispense strategy whereby a wash buffer is added to the reservoir to dilute the contents, magnetically responsive beads are localized within the reservoir with a magnet and most of the solution is dispensed from the reservoir, and this cycle is repeated till acceptable levels of washing are achieved.

As an example, washing magnetically responsive beads may generally include the following steps:

-   -   (1) providing a droplet comprising magnetically responsive beads         and unbound substances in proximity with a magnet;     -   (2) using droplet operations to combine a wash droplet with the         droplet comprising the magnetically responsive beads;     -   (3) immobilizing the beads by application of a magnetic field;     -   (4) using droplet operations to remove some or all of the         droplet surrounding the beads to yield a droplet comprising the         beads with a reduced concentration of unbound components and a         droplet comprising unbound components;     -   (5) releasing the beads by removing the magnetic field; and     -   (6) repeating steps (2) to (3) or (2) to (4) until a         predetermined degree of purification is achieved.

In this manner, unbound substances, such as contaminants, byproducts or excess reagents, can be separated from the beads. Each cycle produces a droplet including the beads but with a decreased level of the unwanted substances. Step (5) is not required in each washing cycle; however, it may be useful to enhance washing by freeing contaminants which may be trapped in the immobilized beads. Steps may be performed in a different order, e.g., steps (2) and (3) may be reversed. Steps in the washing protocol may be accomplished on a droplet actuator using droplet operations as described herein.

In embodiments in which magnetically responsive beads are used, the inventors have found that application of a magnetic field though useful for temporarily immobilizing beads, moving beads and/or positioning beads, sometimes results in unwanted aggregation of the beads. As already noted, in one embodiment, a hydrophilic polymer and/or surfactant is included to prevent or reduce bead aggregation. Hydrophilic polymers and surfactants should be selected and used in amounts which reduce or eliminate bead aggregation and minimize non-specific adsorption while at the same time not resulting in significant loss of target analytes or reagents from the droplet. In one embodiment, the hydrophilic polymer and/or surfactant reduces bead clumping in a droplet in a non-gaseous filler fluid and specifically does not serve to reduce molecular adsorption of droplet components to a surface of the droplet actuator.

Another approach to eliminating or reducing clumping aggregation of beads involves the use of smaller numbers of larger beads. Any number of beads which can be contained in a droplet during one or more droplet operations may be used. In some embodiments, the number of magnetically responsive beads can range from 1 to several 100,000's. For example, in one embodiment, the invention makes use of one to 100 magnetically responsive beads per droplet. For example, the invention may make use of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . 100 magnetically responsive beads per droplet. In one embodiment, the number of magnetically responsive beads is from one to 10. Use of smaller numbers of magnetically responsive beads permits larger beads to be used. For example, in one embodiment, the invention makes use of one to 100 magnetically responsive beads per droplet, where the beads have an average diameter of about 25 to about 100 microns. In another embodiment the invention makes use of one to 10 magnetically responsive beads per droplet, where the beads have an average diameter of about 50 to about 100 microns.

9 CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the invention. Other embodiments having different structures and operations do not depart from the scope of the present invention.

This specification is divided into sections for the convenience of the reader only. Headings should not be construed as limiting of the scope of the invention.

It will be understood that various details of the present invention may be changed without departing from the scope of the present invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation, as the present invention is defined by the claims as set forth hereinafter. 

1. A method of providing a droplet in contact with a magnetically responsive bead and having a reduced quantity of a substance, the method comprising: (a) providing a droplet actuator comprising: (i) a substrate comprising electrodes arranged for conducting droplet operations on a surface; and (ii) a starting droplet comprising: (1) one or more magnetically responsive beads; (2) a starting quantity of the substance; and (3) a starting volume; (b) magnetically immobilizing the one or more magnetically responsive beads at a location which is at a distance from a target droplet splitting zone; and (c) conducting one or more droplet operations comprising droplet dividing operations selected to divide the combined droplet to yield a set of droplets comprising: (i) a droplet comprising substantially all of the one or more magnetically responsive beads and having a decreased quantity of the substance relative to the starting concentration; and (ii) a droplet substantially lacking the magnetically responsive beads.
 2. The method of claim 1 wherein the starting droplet comprises a starting concentration of the substance, and the one or more droplet operations yield a droplet comprising a reduced concentration and reduced quantity of the substance relative to the starting concentration and starting quantity.
 3. The method of claim 1 further comprising conducting one or more droplet operations to merge a wash droplet with the droplet provided in step 1(a) to yield a combined droplet.
 4. The method of claim 1 wherein the one or more magnetically responsive beads comprises one or more beads having a diameter ranging from about 0.1 nm to about 25 mm.
 5. The method of claim 1 further comprising releasing or resuspending one or more of the beads following step 1(c).
 6. The method of claim 5 further comprising providing a hydrophilic polymer in the droplet that improves resuspension relative to a corresponding droplet lacking the hydrophilic polymer.
 7. The method of claim 5 further comprising providing a surfactant in the droplet that improves resuspension relative to a corresponding droplet lacking the hydrophilic polymer.
 8. The method of claim 5 further comprising providing a surfactant in the droplet that: (a) reduces bead clumping in the droplet in a non-gaseous filler fluid; and (b) does not serve to reduce molecular adsorption of droplet components to a surface of the droplet actuator.
 9. The method of claim 5 further comprising agitating one or more of the beads following step 1(c) using a sonicator.
 10. The method of claim 1 wherein: (a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and (b) the strength and location of the magnetic field are selected to immobilize magnetically responsive beads in the droplet at a distance from a target splitting zone which is sufficient to retain substantially all of the magnetic beads in one droplet.
 11. The method of claim 1 wherein: (a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and (b) the location of the source of the magnetic field is selected to centrally locate the magnetic beads along a lateral dimension of the droplet.
 12. The method of claim 1 wherein: (a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and (b) the location of the source of the magnetic field is selected to centrally locate the magnetic beads along a vertical dimension of the droplet.
 13. The method of claim 1 wherein: (a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and (b) the location of the source of the magnetic field is selected to centrally locate the magnetic beads along: (i) a vertical dimension of the droplet; and (ii) a lateral dimension of the droplet.
 14. The method of claim 1 wherein step 1(b) comprises magnetically immobilizing the one or more magnetically responsive beads using a means comprising: (a) an electromagnet; and (b) a means for controlling power supply to the electromagnet.
 15. The method of claim 1 wherein step 1(b) comprises magnetically immobilizing the one or more magnetically responsive beads using a means comprising: (a) means for effecting a magnetic field comprises a magnet; and (b) a means for moving the magnet into and out of proximity with the one or more of the electrodes.
 16. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99% of beads present in the combined droplet.
 17. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99.9% of beads present in the combined droplet.
 18. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99.999% of beads present in the combined droplet.
 19. The method of claim 1 wherein step 1(c) is electrode-mediated.
 20. The method of claim 1 wherein step 1(c) is electrowetting-mediated.
 21. The method of claim 1 wherein step 1(c) is dielectrophoresis-mediated.
 22. The method of claim 1 wherein step 1(c) is electric field-mediated.
 23. The method of claim 1 wherein the substance comprises one or more contaminants.
 24. The method of claim 1 wherein the substance comprises one or more analytes.
 25. The method of claim 1 wherein the magnetically responsive bead comprises a first component having specific or non-specific affinity for a second component provided in the droplet of 1(a).
 26. The method of claim 1 wherein the beads comprise one or more populations of pre-selected biological cells.
 27. The method of claim 25 wherein the affinity comprises an affinity for non-specific adsorption.
 28. The method of claim 25 wherein the affinity comprises an affinity for specific adsorption.
 29. The method of claim 25 wherein the first component comprises an antigen binding region.
 30. The method of claim 25 wherein the first component comprises an antibody binding region.
 31. The method of claim 25 wherein the first component comprises an antibody.
 32. The method of claim 25 wherein the first component comprises two or more antibodies, each having affinity for a different second component.
 33. The method of claim 25 wherein the first component comprises an analyte.
 34. The method of claim 25 wherein the first component comprises two or more analytes, each having affinity for a different second component.
 35. The method of claim 25 wherein the first component comprises an antigen.
 36. The method of claim 25 wherein the first component comprises a nucleic acid.
 37. The method of claim 25 wherein the first component comprises a biotin compound or streptavidin compound.
 38. The method of claim 25 wherein the second component comprises an antigen binding region.
 39. The method of claim 25 wherein the second component comprises an antibody.
 40. The method of claim 25 wherein the second component comprises two or more antibodies, each having affinity for a different type of second component.
 41. The method of claim 25 wherein the second component comprises an analyte.
 42. The method of claim 25 wherein the second component comprises two or more analytes, each having affinity for a different type of second component.
 43. The method of claim 25 wherein the second component comprises an antigen.
 44. The method of claim 25 wherein the second component comprises a nucleic acid.
 45. The method of claim 25 wherein the second component comprises amplified nucleic acid.
 46. The method of claim 25 wherein the second component comprises a biotin compound or streptavidin compound.
 47. The method of claim 25 wherein the second component comprises a contaminant
 48. The method of claim 25 wherein: (a) the first component comprises a compound which binds to the second component; and (b) the second component comprises a compound that binds to the first component.
 49. The method of claim 1 wherein the beads comprise biological cells bound thereto.
 50. The method of claim 1 wherein the beads comprise one or more populations of pre-selected biological cells.
 51. The method of claim 49 wherein the droplet comprises culture medium selected for growing the biological cells.
 52. The method of claim 51 further comprising using the droplet substantially lacking the magnetically responsive beads for conducting one or more droplet operations in an assay protocol.
 53. The method of claim 51 wherein the beads comprise a substantially pure population of biological cells bound thereto.
 54. The method of claim 51 wherein the beads comprise interacting populations of cells.
 55. The method of claim 1 wherein the droplet substantially lacking the magnetically responsive beads comprises one or more beads that are not substantially magnetically responsive.
 56. The method of claim 55 wherein the one or more beads that are not substantially magnetically responsive are bound to one or more second target substances.
 57. A method of providing a nutrient to a biological cell, the method comprising: (a) providing a droplet in contact with a magnetically responsive bead according to the method of claim 1, where the beads comprise one or more populations of biological cells; and (b) conducting one or more droplet operations to bring into contact with the magnetically responsive beads a fluid comprising the nutrient.
 58. A method of splitting a first droplet comprising beads, the method comprising: (a) positioning one or more magnets in proximity to the first droplet sufficient to result in a centralized localization of the beads within the droplet; and (b) splitting the droplet to yield one or more droplets comprising beads and one or more droplets substantially lacking beads.
 59. A method of splitting a first droplet comprising beads, the method comprising: (a) providing a droplet comprising: (i) beads; and (ii) an additive selected and provided in an amount sufficient to enhance bead immobilization in the presence of a magnetic field and/or resuspension following removal of a magnetic field relative to a corresponding control droplet lacking such additive; and (b) splitting the droplet to yield one or more droplets comprising beads and one or more droplets substantially lacking beads.
 60. A method of splitting a first droplet comprising beads, the method comprising: (a) providing a magnetic field in proximity to the first droplet sufficient to substantially immobilize the beads within the droplet; and (b) splitting the droplet at a position sufficiently distant from the immobilized beads to yield one or more droplets comprising beads and one or more droplets substantially lacking beads.
 61. A droplet actuator device comprising: (a) an array of electrodes associated with a substrate and configured for conducting one or more droplet operations; (b) a magnet arranged to exert a magnetic field on a droplet positioned on a first set of one or more of the electrodes; and (c) a magnetic shield arranged to shield a droplet positioned on a second set of one or more of the electrodes from the magnetic field.
 62. The droplet actuator device of claim 61 further comprising: (a) a second magnet arranged to exert a magnetic field on a droplet positioned on a second set of one or more of the electrodes; and (b) a magnetic shield, which may be the same or different from the magnetic shield of 61(c), arranged to shield a droplet positioned on the first set of one or more of the electrodes from the magnetic field.
 63. The droplet actuator device of claim 62 comprising a series of magnet sets and magnetic shield sets, wherein: (a) each magnet set comprises one or more magnets; (b) each magnetic shield set comprises one or more magnetic shields; and (c) the magnet sets and magnetic shield sets are arranged to permit independent droplet operations on a series of droplet sets, wherein: (i) each droplet set comprises one or more droplets; and (ii) each droplet set is influenced by a single magnet set and is blocked by one or more magnetic shield sets from influence by other magnet sets.
 64. The droplet actuator device of claim 61 comprising a droplet: (a) comprising one or more magnetically responsive beads thereon; and (b) positioned on one or more electrodes from the first set, so that the magnetically responsive beads are attracted by the magnetic field.
 65. The droplet actuator device of claim 61 comprising a droplet: (a) comprising one or more magnetically responsive beads thereon; and (b) positioned on one or more electrodes from the second set, so that the magnetically responsive beads are not attracted by the magnetic field.
 66. The droplet actuator device of claim 61 wherein the magnetic shield comprises a material having high magnetic permeability.
 67. The droplet actuator device of claim 61 wherein the magnetic shield comprises a material selected from the group consisting of Mu metals, permalloys, iron, steel, nickel, and alloys substantially comprising any of the foregoing.
 68. A method of conducting a droplet operation on a droplet actuator device using a droplet comprising magnetic beads, the method comprising: (a) providing droplet actuator device comprising: (i) an array of electrodes associated with a substrate and configured for conducting one or more droplet operations; (ii) a magnet arranged to exert a magnetic field on a droplet positioned on a first set of one or more of the electrodes; and (iii) a magnetic shield arranged to shield a droplet positioned on a second set of one or more of the electrodes from the magnetic field; (b) positioning a first droplet comprising one or more magnetically responsive beads on one or more electrodes from the first set, so that the magnetically responsive beads are attracted by the magnetic field; (c) positioning a second droplet comprising one or more magnetically responsive beads on one or more electrodes from the second set, so that the magnetically responsive beads are not attracted by the magnetic field; and (d) conducting a droplet operation on the first droplet and/or conducting a droplet operation on the second droplet.
 69. A droplet actuator device comprising magnet sets and electrode sets, wherein: (a) each electrode set comprises one or more electrodes associated with a substrate; (b) two or more electrode sets are: (i) arranged to form an array of electrodes; and (ii) configured for conducting one or more droplet operations; (c) each magnet set comprises one or more magnets; (d) each magnet set is arranged in a relationship with an electrode set such that magnetic field(s) exerted by the magnet set restricts mobility of magnetically responsive particles to a locus which is proximate to the electrode set; and (e) each magnet set is arranged in relationship to its adjacent magnet set(s), which relationship a polar opposite to the polar orientation of the adjacent magnet set.
 70. The droplet actuator device of claim 69 wherein each magnet set is oriented with a pole facing an electrode set, and each adjacent magnet set is oriented with an opposite pole facing the electrode set.
 71. The droplet actuator device of claim 69 wherein each magnet has a sufficiently strong field to immobilize magnetically responsive particles during a splitting operation, but not sufficiently strong to immobilize magnetically responsive particles during a droplet transport operation.
 72. A droplet actuator device comprising: (a) electrodes arranged in association with a substrate to conduct droplet operations on the substrate, the droplet operations comprising droplet splitting operations and droplet transport operations; (b) a magnet associated with a subset or electrodes comprising one or more of the electrodes; and (c) a droplet on one or more electrodes of the subset of electrodes, the droplet comprising one or more magnetically responsive particles or substances; wherein the magnet is oriented to exert a magnetic field in proximity to the droplet, the magnetic field exerting sufficient force on the magnetically responsive particles or substances to immobilize the magnetically responsive particles or substances during a droplet splitting operation, but not sufficiently strong to immobilize magnetically responsive particles during a droplet transport operation.
 73. A method of reducing carryover in a droplet actuator, the method comprising: (a) transporting a droplet comprising a target substance past a locus; (b) transporting a wash droplet not comprising the target substance past the locus; and (c) repeating steps (a) and (b), wherein the target substance may be the same or different.
 74. The method of claim 73 wherein the locus comprises a locus in proximity to a sensor arranged to sense a property of the droplet.
 75. The method of claim 73 wherein the wash droplet comprises a buffer droplet.
 76. A method of eliminating a satellite droplet, the method comprising: (a) conducting a droplet splitting operation at a locus on a droplet in a filler fluid on a droplet actuator, the splitting operation yielding two or more sub-droplets and one or more satellite droplets; (b) conducting a droplet transport operation on one or more of the sub-droplets yielded in step (a) to transport the one or more sub-droplets away from the locus; and (c) transporting a wash droplet into proximity with the locus to merge the one or more of the satellite droplets with the wash droplet.
 77. The method of claim 76 wherein the wash droplet comprises a buffer droplet.
 78. A method of exposing a droplet to a sensor, the method comprising: (a) providing droplet on a substrate in sensing proximity to a sensor, the droplet having a starting surface area exposed to the sensor; and (b) effecting a change in the droplet's shape and producing an expanded surface area of the droplet in sensing proximity to the sensor, wherein the expanded surface area exposed to the sensor is greater than the starting surface area exposed to the sensor.
 79. The method of claim 78 wherein step 78(b) is effected by steps comprising activating one or more electrodes associated with the substrate.
 80. The method of claim 78 wherein step 78(b) is effected by an effect comprising an electrowetting mediated effect.
 81. The method of claim 78 where the sensor comprises a photon sensor, and the droplet comprises a substance that emits photons.
 82. The method of claim 78 where the change in the droplet's shape produces a slug.
 83. The method of claim 78 where the change in the droplet's shape produces a series of droplets.
 84. A method of exposing a surface area of a droplet to a sensor, the method comprising exposing to a sensor a droplet on a substrate, the droplet having an expanded surface area effected at least in part by activation of one or more electrodes associated with the substrate to effect a change in the droplet's shape and producing the expanded surface area. 85-114. (canceled) 